Threads of Hyaluronic acid and/or derivatives thereof, methods of making thereof and uses thereof

ABSTRACT

The present invention provides threads of hyaluronic acid, and/or derivatives thereof, methods of making thereof and uses thereof, for example, in aesthetic applications (e.g., dermal fillers), surgery (sutures), drug delivery, etc.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Phase application under 35 U.S.C. §371 ofPCT Application PCT/US2009/055704, filed Sep. 2, 2009, which claimsbenefit under 35 U.S.C. §119(e) from U.S. Provisional Application Ser.No. 61/190,866, filed Sep. 2, 2008, which are hereby incorporated byreference in their entirety.

FIELD

The present invention relates generally to threads of hyaluronic acid,and/or derivatives thereof, methods of making thereof and uses thereof,for example, in aesthetic applications (e.g., dermal fillers), surgery(e.g., sutures), drug delivery, negative pressure wound therapy, moistwound dressing, etc.

BACKGROUND

Hyaluronic acid is a linear polysaccharide (i.e., non-sulfatedglycosaminoglycan) consisting of a repeated disaccharide unit ofalternately bonded β-D-N-acetylglucoamine and β-D-glucuronic acid (i.e.,(-4GlcUAβ1-3GlcNAcβ1-)_(n)) which is a chief component of theextracellular matrix and is found, for example, in connective,epithelial and neural tissue. Natural hylauronic acid is highlybiocompatible because of its lack of species and organ specificity andthus is often used as a biomaterial in tissue engineering and as acommon ingredient in various dermal fillers.

Various chemically modified forms of hyaluronic acid (e.g., cross linkedforms, ionically modified forms, esterified forms, etc.) have beensynthesized to address a significant problem associated with naturalhyaluronic acid which has poor in vivo stability due to rapid enzymaticdegradation and hydrolysis. Currently, hyaluronic acid or cross linkedversions thereof are used in various gel forms, for example as dermalfillers, adhesion barriers, etc.

However, substantial issues exist with the use of gels of hyaluronicacid or cross linked versions thereof. First, the force required todispense gels of hyaluronic acid or cross linked versions thereof isnon-linear which causes the initial “glob” that many physicians reportwhen injecting hyaluronic acid or cross linked versions thereof. Second,precisely dispensing hyaluronic gels to specific locations is verydifficult because such gels have little mechanical strength. Further,the gel will occupy the space of least resistance which makes its use inmany applications (e.g., treatment of fine wrinkles) problematic.

Accordingly, what is needed are new physical forms of hyaluronic acid orcross linked versions thereof which can be dispensed uniformly tospecific locations regardless of tissue resistance. Such new forms mayhave particular uses, for example, in aesthetic and surgicalapplications, drug delivery, wound therapy and wound dressing.

SUMMARY

The present invention satisfies these and other needs by providing, inone aspect, a thread of hyaluronic acid or salts, hydrates or solvatesthereof and, in a second aspect, a thread of cross linked hyaluronicacid or salts, hydrates or solvates thereof. In some embodiments, thethread is a combination of a thread of hyaluronic acid or salts,hydrates or solvates thereof and a thread of cross linked hyaluronicacid or salts, hydrates or solvates thereof.

In a third aspect, a method of making a thread of hyaluronic acid orsalts, hydrates or solvates thereof is provided. Hyaluronic acid orsalts, hydrates or solvates thereof are mixed with water or a buffer toform a gel. The gel is extruded to form a thread. The thread is thendried to provide a thread of hyaluronic acid.

In a fourth aspect, a method of making a thread of cross linkedhyaluronic acid or salts, hydrates or solvates thereof is provided.Hyaluronic acid or salts, hydrates or solvates thereof are mixed withwater or a buffer and a cross linking agent to form a gel. The gel isextruded to form a thread. The thread is then dried to provide a threadof cross linked hyaluronic acid.

In a fifth aspect a method of treating a wrinkle in a subject in needthereof is provided. A thread of hyaluronic acid or salts, hydrates orsolvates thereof or a thread of cross linked hyaluronic acid or salts,hydrates or solvates thereof or a combination thereof is attached to theproximal aspect of a needle. The distal end of the needle is insertedthrough the skin surface of the subject into the dermis adjacent to orwithin the wrinkle. The dermis of the subject in the base of the wrinkleis traversed with the needle. The needle then exits the skin surface ofthe subject and is pulled distally until it is removed from the skin ofthe subject such that the thread is pulled into the location previouslyoccupied by the needle. The excess thread is cut from the needle at theskin surface of the subject.

In still other aspects, methods of using threads of hyaluronic acid orsalts, hydrates or solvates thereof or threads of cross linkedhyaluronic acid or salts, hydrates or solvates thereof or combinationsthereof, for example, as dermal fillers, adhesion barriers, wounddressings including negative pressure wound dressings, sutures, etc. isprovided. Further provided are methods of using threads of hyaluronicacid or salts, hydrates or solvates thereof or threads of cross linkedhyaluronic acid or salts, hydrates or solvates thereof or combinationsthereof, for example, in surgery, opthamology, wound closure, drugdelivery, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a thread attached to the proximal end of a needle, inits entirety;

FIG. 2A illustrates a close-up view of a thread inserted into theinner-diameter of a needle;

FIG. 2B illustrates a close-up view of the proximal end of a solidneedle with the thread overlapping the needle;

FIG. 3A illustrates a fine, facial wrinkle in the peri-orbital region ofa human;

FIG. 3B illustrates a needle and thread being inserted into the dermisof the wrinkle at the medial margin;

FIG. 3C illustrates the needle being adjusted to traverse beneath thewrinkle;

FIG. 3D illustrates the needle exiting at the lateral margin of thewrinkle;

FIG. 3E illustrates the needle having pulled the thread into thelocation it previously occupied beneath the wrinkle;

FIG. 3F illustrates the thread implanted beneath the wrinkle, withexcess thread having been cut off;

FIG. 4A illustrates a top-down view of a male with typical male-patternbaldness;

FIG. 4B illustrates where hair re-growth is desired, taking hair-linesinto consideration;

FIG. 4C illustrates a curved needle with attached thread being insertedinto one imaginary line where hair re-growth is desired;

FIG. 4D illustrates the needle traversing the imaginary line, andexiting the skin;

FIG. 4E illustrates the needle pulled through distally, pulling alongthe thread into the desired location;

FIG. 4F illustrates scissors being used to cut excess thread;

FIG. 5A illustrates a cross-sectional view of a fold or a wrinkle;

FIG. 5B illustrates a thread implanted beneath a wrinkle that is not yethydrated;

FIG. 5C illustrates a thread implanted beneath a wrinkle that is fullyhydrated and has flattened the surface appearance of the wrinkle;

FIG. 6A illustrates a human pancreas with a tumor;

FIG. 6B illustrates a curved needle with a thread attached thereto;

FIG. 6C illustrates a curved needle traversing the tumor within thepancreas;

FIG. 6D illustrates the end-result of repeated implantations of thread;

FIG. 7A illustrates multiple layers of concentric coils of thread,shaped to represent a human nipple;

FIG. 7B illustrates the implant of FIG. 7A in cross-section;

FIG. 7C illustrates how an implant of coiled thread would be used fornipple reconstruction; and

FIG. 8 illustrates how a needle and thread could be used to place athread in a specific, linear location to promote nerve or vesselregrowth in a specific line.

DETAILED DESCRIPTION

Definitions

“Buffer” includes, but is not limited to,2-amino-2-methyl-1,3-propanediol, 2-amino-2-methyl-1-propanol,L-(+)-tartaric acid, D-(−)-tartaric acid, ACES, ADA, acetic acid,ammonium acetate, ammonium bicarbonate, ammonium citrate, ammoniumformate, ammonium oxalate, ammonium phosphate, ammonium sodiumphosphate, ammonium sulfate, ammonium tartrate, BES, BICINE, BIS-TRIS,bicarbonate, boric acid, CAPS, CHES, calcium acetate, calcium carbonate,calcium citrate, citrate, citric acid, diethanolamine, EPP,ethylenediaminetetraacetic acid disodium salt, formic acid solution,Gly-Gly-Gly, Gly-Gly, glycine, HEPES, imidazole, lithium acetate,lithium citrate, MES, MOPS, magnesium acetate, magnesium citrate,magnesium formate, magnesium phosphate, oxalic acid, PIPES, phosphatebuffered saline, phosphate buffered saline, piperazine potassiumD-tartrate, potassium acetate, potassium bicarbonate, potassiumcarbonate, potassium chloride, potassium citrate, potassium formate,potassium oxalate, potassium phosphate, potassium phthalate, potassiumsodium tartrate, potassium tetraborate, potassium tetraoxalatedehydrate, propionic acid solution, STE buffer solution, sodium5,5-diethylbarbiturate, sodium acetate, sodium bicarbonate, sodiumbitartrate monohydrate, sodium carbonate, sodium citrate, sodiumformate, sodium oxalate, sodium phosphate, sodium pyrophosphate, sodiumtartrate, sodium tetraborate, TAPS, TES, TNT, TRIS-glycine,TRIS-acetate, TRIS buffered saline, TRIS-HCl, TRIS phosphate-EDTA,tricine, triethanolamine, triethylamine, triethylammonium acetate,triethylammonium phosphate, trimethylammonium acetate, trimethylammoniumphosphate, Trizma® acetate, Trizma® base, Trizma® carbonate, Trizma®hydrochloride or Trizma® maleate.

“Salt” refers to a salt of hyaluronic acid, which possesses the desiredactivity of the parent compound. Such salts include, but are not limitedto: (1) acid addition salts, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, t-butylacetic acid, laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid,salicylic acid, stearic acid, muconic acid and the like; or (2) saltsformed when an acidic proton present in the parent compound is replacedby an ammonium ion, a metal ion, e.g., an alkali metal ion (e.g., sodiumor potassium), an alkaline earth ion (e.g., calcium or magnesium), or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, N-methylglucamine, morpholine,piperidine, dimethylamine, diethylamine and the like. Also included aresalts of amino acids such as arginates and the like, and salts oforganic acids like glucurmic or galactunoric acids and the like.

Threads of Hyaluronic Acid and Derivatives Thereof

The present invention generally provides threads of hyaluronic acid orsalts, hydrates or solvates thereof, threads of cross linked hyaluronicacid or salts, hydrates or solvates thereof and combinations thereof. Insome embodiments, the hyaluronic acid is isolated from an animal source.In other embodiments, the hyaluronic acid is isolated from bacterialfermentation

In some embodiments, the lifetime of the threads of hyaluronic acid orsalts, hydrates or solvates thereof, in vivo is between about 1 minuteand about 1 month. In other embodiments, the lifetime of the thread ofhyaluronic acid or salts, hydrates or solvates thereof, in vivo isbetween about 10 minutes and about 1 week. In still other embodiments,the lifetime of the thread of hyaluronic acid or salts, hydrates orsolvates thereof, in vivo is between about 1 hour and about 3 days.

In some embodiments, the lifetime of the thread of cross linkedhyaluronic acid or salts, hydrates or solvates thereof, in vivo isbetween about 1 week and about 24 months. In other embodiments, thelifetime of the thread of cross linked hyaluronic acid or salts,hydrates or solvates thereof, in vivo is between about 1 month and about12 months. In still other embodiments, the lifetime of the thread ofhyaluronic acid or salts, hydrates or solvates thereof, in vivo isbetween about 3 months and about 9 months.

In some embodiments, hyaluronic acid or salts, hydrates or solvatesthereof have been cross linked with butanediol diglycidyl ether (BDDE),divinyl sulfone (DVS) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC). Those of skill in the art will appreciate that manyother cross linking agents may be used to crosslink hyaluronic acid orsalts, hydrates or solvates thereof. Accordingly, the above list ofcross linking agents is illustrative rather than comprehensive.

In some of the above embodiments, the degree of cross linking betweenhyaluronic acid or salts, hydrates or solvates thereof and the crosslinking agent is between about 0.01% and about 20%. In other of theabove embodiments, the degree of cross linking between hyaluronic acidor salts, hydrates or solvates thereof and the cross linking agent isbetween about 0.1% and about 10%. In still other of the aboveembodiments, the degree of cross linking between hyaluronic acid orsalts, hydrates or solvates thereof and the cross linking agent isbetween about 1% and about 8%.

In some of the above embodiments, the thread includes one or moretherapeutic or diagnostic agents. In other of the above embodiments, thediagnostic agent is soluble TB (tuberculosis) protein. In still other ofthe above embodiments, the therapeutic agent is an anesthetic, includingbut not limited to, lidocaine, xylocaine, novocaine, benzocaine,prilocaine, ripivacaine, propofol or combinations thereof. In stillother of the above embodiments, the therapeutic agent is epinephrine,adrenaline, ephedrine, aminophylline, theophylline or combinationsthereof. In still other of the above embodiments, the therapeutic agentis botulism toxin. In still other of the above embodiments, thetherapeutic agent is laminin-511. In still other of the aboveembodiments, the therapeutic agent is glucosamine, which can be used,for example, in the treatment of regenerative joint disease. In stillother of the above embodiments, the therapeutic agent is an antioxidant,including but not limited to, vitamin E or all-trans retinoic acid suchas retinol. In still other of the above embodiments, the therapeuticagent includes stem cells. In still other of the above embodiments, thetherapeutic agent is insulin, a growth factor such as, for example, NGF(nerve growth factor), BDNF (brain-derived neurotrophic factor), PDGF(platelet-derived growth factor) or Purmorphamine Deferoxamine NGF(nerve growth factor), dexamethasone, ascorbic acid, 5-azacytidine,4,6-disubstituted pyrrolopyrimidine, cardiogenols, cDNA, DNA, RNAi,BMP-4 (bone morphogenetic protein-4), BMP-2 (bone morphogeneticprotein-2), an antibiotic agent such as, for example, β lactams,quinolones including fluoroquinolones, aminoglycosides or macrolides, ananti-fibrotic agent, including but not limited to, hepatocyte growthfactor or Pirfenidone, an anti-scarring agent, such as, for example,anti-TGF-b2 monoclonal antibody (rhAnti-TGF-b2 mAb), a peptide such as,for example, GHK copper binding peptide, a tissue regeneration agent, asteroid, fibronectin, a cytokine, an analgesic such as, for example,Tapentadol HCl, opiates, (e.g., morphine, codone, oxycodone, etc.) anantiseptic, alpha- beta or gamma-interferon, EPO, glucagons, calcitonin,heparin, interleukin-1, interleukin-2, filgrastim, a protein, HGH,luteinizing hormone, atrial natriuretic factor, Factor VIII, Factor IX,or a follicle-stimulating hormone. In still other of the aboveembodiments, the thread contains a combination of more than onetherapeutic agent or diagnostic agent. In some of these embodiments,different threads comprise different therapeutic agents or diagnosticagents.

In some of the above embodiments, the thread has an ultimate tensilestrength of between about 0 kpsi and about 250 kpsi. In other of theabove embodiments, the thread has an ultimate tensile strength ofbetween about 1 kpsi and about 125 kpsi. In still other of the aboveembodiments, the thread has an ultimate tensile strength of betweenabout 5 kpsi and about 100 kpsi.

In some of the above embodiments, the thread has an axial tensilestrength of between about 0.01 lbs and about 10 lbs. In other of theabove embodiments, the thread has an axial tensile strength of betweenabout 0.1 lbs and about 5 lbs. In still other of the above embodiments,the thread has an axial tensile strength of between about 0.5 lbs andabout 2 lbs.

In some of the above embodiments, the thread has a cross-section area ofbetween about 1*10⁶ in² and about 1,000*10⁶ in². In other of the aboveembodiments, the thread has a cross-section area of between about 10*10⁶in² and about 500*10⁶ in². In still other of the above embodiments, thethread has a cross-section area of between about 50*10⁶ in² and about250*10⁶ in².

In some of the above embodiments, the thread has a diameter of betweenabout 0.0001 in and about 0.100 in. In other of the above embodiments,the thread has a diameter of between about 0.001 in and about 0.020 in.In still other of the above embodiments, the thread has a diameter ofbetween about 0.003 and about 0.010 in.

In some of the above embodiments, the thread has an elasticity ofbetween about 1% and 200%. In other of the above embodiments, the threadhas an elasticity of between about 5% and about 100%. In still other ofthe above embodiments, the thread has an elasticity of between about 10%and 50%. Herein, elasticity is the % elongation of the thread whileretaining ability to return to the initial length of the thread.

In some of the above embodiments, the thread has a molecular weight ofbetween about 0.1 MD and about 8 MD (MD is a million Daltons). In otherof the above embodiments, the thread has a molecular weight of betweenabout 0.5 MD to about 4 MD. In still other of the above embodiments, thethread has a molecular weight of between about 1 MD to about 2 MD.

In some of the above embodiments, the thread has a persistent chainlength of between about 10 nm and about 250 nm. In other of the aboveembodiments, the thread has a persistent chain length of between about10 nm and about 125 nm. In still other of the above embodiments, thethread has a persistent chain length of between about 10 nm and about 75nm.

In some of the above embodiments, the cross-sectional area of the threadwhen fully hydrated swells to between about 0% to about 10,000%. Inother of the above embodiments, the cross-sectional area of the threadwhen fully hydrated swells to between about 0% to about 2,500%. In stillother of the above embodiments, the cross-sectional area of the threadwhen fully hydrated swells to between about 0% to about 900%.

In some of the above embodiments, the thread elongates when fullyhydrated to between about 0% to about 1,000%. In other of the aboveembodiments, the thread elongates when fully hydrated to between about0% to about 100%. In still other of the above embodiments, the threadelongates when fully hydrated to between about 0% to about 30%.

In some of the above embodiments, the thread is fully hydrated aftersubmersion in an aqueous environment in between about 1 second and about24 hours. In other of the above embodiments, the thread is fullyhydrated after submersion in an aqueous environment in between about 1second and about 1 hour. In still other of the above embodiments, thethread is fully hydrated after submersion in an aqueous environment inbetween about 1 second to about 5 minutes.

In some embodiments, the thread is cross linked and has an ultimatetensile strength of between about 50 kpsi and about 75 kpsi, a diameterof between 0.005 in and about 0.015 in, the thickness or diameter of thethread when fully hydrated swells between about 50% to about 100% andthe lifetime of the thread in vivo is about 6 months.

In some embodiments, braids may be formed from the threads describedabove. In other embodiments, cords may be formed from the threadsdescribed above. In still other embodiments, a woven mesh may be formedfrom the threads described above. In still other embodiments, a wovenmesh may be formed from the braids or cords described above.

In some embodiments, a three-dimensional structure may be constructed byweaving or wrapping or coiling or layering the threads described above.In other embodiments, a three-dimensional structure may be constructedby weaving or wrapping or coiling or layering the braids describedabove. In still other embodiments, a three-dimensional structure may beconstructed by weaving or wrapping or coiling or layering the cordsdescribed above. In still other embodiments, a three-dimensionalstructure may be constructed by weaving or wrapping or coiling orlayering the meshes described above.

In some embodiments, a three-dimensional, cylindrical implant is made ofany of the threads is provided. An exemplary use for such an implant isfor nipple reconstruction. In some embodiments, the threads used to makethe cylindrical implant are cross linked and include chondrocyteadhesion compounds. In other embodiments, the cylindrical shape isprovided by multiple, concentric coils of threads.

Threads of hyaluronic acid and/or derivatives thereof may contain one ormore chiral centers and therefore, may exist as stereoisomers, such asenantiomers or diastereomers. In general, all stereoisomers (i.e., allpossible enantiomers and stereoisomers of the illustrated compoundsincluding the stereoisomerically pure form (e.g., enantiomerically pureor diastereomerically pure) and enantiomeric and stereoisomeric mixturesare within the scope of the present invention.

Threads of hyaluronic acid and/or derivatives thereof may exist inseveral tautomeric forms and mixtures thereof all of which are withinthe scope of the present invention. Threads of hyaluronic acid and/orderivatives thereof may exist in unsolvated forms as well as solvatedforms, including hydrated forms. In general, hydrated and solvated formsare within the scope of the present invention. Accordingly, all physicalforms of threads of hyaluronic acid and/or derivatives thereof areequivalent for the uses contemplated by the present invention and areintended to be within the scope of the present invention.

Methods of Making Threads of Hyaluronic Acid and Derivatives Thereof

The present invention also provides methods for making threads ofhyaluronic acid and derivatives thereof as described above. In someembodiments, a method of making threads of hyaluronic acid or salts,hydrates or solvates thereof, is provided Hyaluronic acid or salts,hydrates or solvates thereof are mixed with water or a buffer to form agel. The gel is then extruded to form a thread of gel. The gel can beextruded, for example, by placing the gel in a syringe with a nozzle,pressurizing the syringe, and linearly translating the syringe as gel isextruded from the nozzle. Nozzle characteristics such as taper, lengthand diameter, the syringe pressure, and the speed of linear translationmay be adjusted to make threads of different sizes and mechanicalcharacteristics. Another method of making a thread of gel is by rollingthe gel, i.e., like dough, or by placing it into a mold. Still anothermethod of making a thread of gel is to allow the gel to stretch into athread under the influence of gravity or using centrifugal force. Stillanother method of making a thread of gel is by shearing the gel inbetween charged parallel glass plates. Yet another method of making athread of gel is by confining the gel into a groove patterned on anelastomer and then stretching the elastomer. Yet another method ofmaking a thread of gel is by confining the gel into a permeable tubularstructure that allows dehydration of the thread, and if necessarycontrolling the nature of the dehydration by adjusting environmentalparameters such as temperature, pressure and gaseous composition. Thethread of hyaluronic acid or salts, hydrates or solvates thereof is thendried after preparation.

In other embodiments, a method of making threads of cross linkedhyaluronic acid or salts, hydrates or solvates thereof, is provided.Hyaluronic acid or salts, hydrates or solvates thereof are mixed withwater or a buffer and a cross linking agent to form a gel. The gel isthen extruded to form a thread as described above or the thread can bemade by any of the methods described above. Generally, the gel should beextruded or otherwise manipulated soon after addition of the crosslinking agent so that cross linking occurs as the thread dries. Thethread of cross linked hyaluronic acid or salts, hydrates or solvatesthereof is then dried after preparation.

In some embodiments, the ratio of cross linking agent to hyaluronic acidis between about 0.01% and about 10%. In other embodiments, the ratio ofcross linking agent to hyaluronic acid is between about 0.02% and about5%. In still other embodiments, the ratio of cross linking agent tohyaluronic acid is between about 0.1% and about 3%.

In some of the above embodiments, one or more therapeutic or diagnosticagents are included in the gel forming step.

In some of the above embodiments, the gel has a concentration by weightof hyaluronic acid of between about 0.1% and about 10%. In other of theabove embodiments, the gel has a concentration by weight of hyaluronicacid of between about 1% and about 7%. In still other of the aboveembodiments, the gel has a concentration by weight of hyaluronic acid ofbetween about 4% and about 6%.

In some of the above embodiments, the polymer chains are furtheroriented along the axis of the thread by being stretched axially. Inother of the above embodiments, the polymer chains are oriented alongthe axis of the thread by gravimetric force or centrifugal force. Instill other of the above embodiments, gravimetric force is applied byhanging the thread vertically. In still other of the above embodiments,the polymer chains are oriented along the axis of the thread byapplication of an electric potential along the length of the thread. Instill other of the above embodiments, the polymer chains are orientedalong the axis of the thread by a combination of the above methods.

In some of the above embodiments, the threads are hydrated with waterand then dried again. In other of the above embodiments, the hydrationand drying steps are repeated multiple times. In still other of theabove embodiments, the polymer chains are oriented along the axis of thethread by being stretched axially, by application of gravimetric forceor centrifugal force, by application of an electric potential along thelength of the thread or by combinations thereof. In still other of theabove embodiments, a therapeutic agent or a diagnostic agent or a crosslinking agent is applied to the thread during the hydration step.

In some of the above embodiments, the gel is extruded over a previouslymade thread to provide a layered thread.

In another of the above embodiments, after the drying step, the threadis submerged or rinsed with an agent. In some of the above embodiments,the agent is a cross linking agent, therapeutic or diagnostic agent.

In another of the above embodiments, while the thread is hydrated, forexample after a rinsing step, the thread is submerged or rinsed with anagent. In some of the above embodiments, the agent is a cross linkingagent, therapeutic or diagnostic agent.

In still other of the above embodiments, the thread is frozen and thenthawed. In still other of the above embodiments, the thread is frozenand then thawed at least more than once.

In still other of the above embodiments, a dried thread is irradiated topromote cross linking. In some of the above embodiments, a hydratedthread is irradiated to promote cross linking.

In still other of the above embodiments, a dried or hydrated thread iscoated to alter the properties of the thread, with a bioabsorbablebiopolymer, such as for example, collagen, PEG or PLGA. Alternatively,woven constructs, whether single layer or 3D, can be coated in theirentirety to create weaves or meshes with altered physical propertiesfrom that of a free-woven mesh.

Methods of Using Threads of Hyaluronic Acid and Derivatives Thereof

The threads, braids, cords, woven meshes or three-dimensional structuresdescribed herein can be used, for example, to fill aneurysms, occludeblood flow to tumors, (i.e., tumor occlusion), in eye-lid surgery, inpenile augmentation (e.g., for enlargement or for sensitivity reduction,i.e., pre-mature ejaculation treatment), inter-nasal (blood-brainbarrier) delivery devices for diagnostic and/or therapeutic agents,corneal implants for drug delivery, nose augmentation or reconstruction,lip augmentation or reconstruction, facial augmentation orreconstruction, ear lobe augmentation or reconstruction, spinal implants(e.g., to support a bulging disc), root canal filler (medicated withtherapeutic agent), glottal insufficiency, laser photo-refractivetherapy (e.g., hyaluronic acid thread/weave used as a cushion),scaffolding for organ regrowth, spinal cord treatment (BDNF and NGF), inParkinson's disease (stereotactic delivery), precise delivery oftherapeutic or diagnostic molecules, in pulp implantation, replacementpulp root canal treatment, shaped root canal system, negative pressurewound therapy, adhesion barriers and wound dressings.

In some embodiments, the threads, braids, cords, woven meshes orthree-dimensional structures described herein are used as dermal fillersin various aesthetic applications. In other embodiments, the threads,braids, cords, woven meshes or three-dimensional structures describedherein are used as sutures in various surgical applications. In stillother embodiments, the threads, braids, cords, woven meshes orthree-dimensional structures described herein are used in ophthalmologicsurgery, drug delivery and intra-articular injection.

In some embodiments, the threads, braids, cords, woven meshes orthree-dimensional structures described herein are used in wounddressings including negative pressure wound dressings.

In some embodiments, wound dressing remains in contact with the woundfor at least 72 hours. In other embodiments, the negative pressure wounddressing remains in contact with the wound for at least 1 week. In stillother embodiments, the wound dressing remains in contact with the woundfor at least 2 weeks. In still other embodiments, the wound dressingremains in contact with the wound for at least 3 weeks. In still otherembodiments, the wound dressing remains in contact with the wound for atleast 4 weeks. In the above embodiments, it should be understood thatgranulation tissue is not retaining the threads, braids, cords, wovenmeshes or three-dimensional structures described herein as thesecomponents are fully absorbable. In some of these embodiments, the wounddressing is between about 1 cm and about 5 cm thick. Accordingly, insome of these embodiments, wound bed closure may be achieved withoutchanging the dressing.

In some embodiments, the woven meshes described herein are used in wounddressings including negative pressure wound dressings. In otherembodiments, the dressing include between 2 and about 10 layers of wovenmeshes.

In still other embodiments, the woven meshes comprise identical threads.In still other embodiments, the woven meshes comprise different threads.

In some embodiments, the woven meshes are between about 1 mm and about 2mm thick when dry. In other embodiments, the woven meshes are betweenabout 2 mm and about 4 mm thick when dry.

In some embodiments, the pore size of the woven mesh is between about 1mm and about 10 mm in width. In other embodiments, the pore size of thewoven mesh is between about 0.3 mm and about 0.6 mm in width. In stillother embodiments, the pores of the woven mesh are aligned. In stillother embodiments, the pores of the woven mesh are staggered. In stillother embodiments, the woven meshes are collimated to create pores ofdesired size.

In some embodiments, the woven mesh is mechanically stable at a vacuumup to about 75 mm Hg. In other embodiments, the woven mesh ismechanically stable at a vacuum up to about 150 mm Hg.

In some embodiments, the woven mesh includes collagen. In otherembodiments, the dressing is attached to a polyurethane foam. In stillother embodiments, the polyurethane foam is open celled. In still otherembodiments, the dressing is attached to a thin film. In still otherembodiments, the thin film is silicone or polyurethane. In still otherembodiments, the dressing is attached to the thin film with a watersoluble adhesive.

In some embodiments, the thread used in the dressing includes atherapeutic agent or a diagnostic agent.

In some embodiments, a negative pressure wound dressing (Johnson et al.,U.S. Pat. No. 7,070,584, Kemp et al., U.S. Pat. No. 5,256,418, Chatelieret al., U.S. Pat. No. 5,449,383, Bennet et al., U.S. Pat. No. 5,578,662,Yasukawa et al., U.S. Pat. Nos. 5,629,186 and 5,780,281 and Ser. No.08/951,832) is provided for use in vacuum induced healing of wounds,particularly open surface wounds (Zamierski U.S. Pat. Nos. 4,969,880,5,100,396, 5,261,893, 5,527,293 and 6,071,267 and Argenta et al., U.S.Pat. Nos. 5,636,643 and 5,645,081). The dressing includes a pad whichconforms to the wound location, an air-tight seal which is removablyadhered to the pad, a negative pressure source in fluid communicationwith the pad and the threads, braids, cords, woven meshes orthree-dimensional structures described herein attached to the woundcontacting surface of the pad. The pad, seal and vacuum source areimplemented as described in the prior art.

In other embodiments, the threads, braids, cords, woven meshes orthree-dimensional structures described herein are mechanically stable ata vacuum up to about 75 mm Hg. In still other embodiments, the threads,braids, cords, woven meshes or three-dimensional structures describedherein are mechanically stable at a vacuum up to about 150 mm Hg. Instill other embodiments, the dressing includes at least one layer ofwoven mesh. In still other embodiments, the dressing include between 2and about 10 layers of woven mesh. In still other embodiments, the padis a foam. In still other embodiments, the pad is an open cellpolyurethane foam.

In some embodiments a tube connects the pad to the negative pressuresource. In still other embodiments, a removable canister is insertedbetween the pad and the negative pressure source and is in fluidcommunication with both the pad and the negative pressure source.

In some embodiments, the threads, braids, cords, woven meshes orthree-dimensional structures described herein are not hydrated.Accordingly, in these embodiments, the dressing could absorb woundexudates when placed in contact with the wound. In other embodiments,the threads, braids, cords, woven meshes or three-dimensional structuresdescribed herein are hydrated. Accordingly, in these embodiments, thedressing could keep the wound moist when placed in contact with thewound.

In some embodiments, an input port attached to a fluid is connected withthe pad. Accordingly, in these embodiments, fluid could be dispensed inthe wound. In some embodiments, the fluid is saline. In otherembodiments, the fluid contains diagnostic or therapeutic agents.

In some embodiments, the threads, braids, cords, woven meshes orthree-dimensional structures described herein are used as adhesionbarriers. In some embodiments, the woven meshes described herein areused in adhesion barriers.

In some embodiments, a method of treating a wrinkle in a subject isprovided. For example, the wrinkle may be in the peri-orbital region asillustrated in FIG. 3A. The thread may be attached to a needle asillustrated, for example, in FIGS. 1, 2A and 2B. The distal end of theneedle may be inserted through the skin surface of the subject into thedermis adjacent to or within the wrinkle as illustrated, for example, inFIG. 3B. In some embodiments, the thread is inserted into thesubcutaneous space instead of the dermis. The needle then may traversethe dermis of the subject beneath the wrinkle as illustrated, forexample, in FIG. 3C. The needle then may exit the skin of the subject atthe opposite margin of the wrinkle, as illustrated, for example, in FIG.3D. The needle may then be pulled distally until it is removed from thesubject such that the thread is pulled into the location previouslyoccupied by the needle beneath the wrinkle, as illustrated, for example,in FIG. 3E. Finally, excess thread is cut from the needle at the skinsurface of the subject which leaves the thread implanted as illustrated,for example, in FIG. 3F.

While not wishing to be bound by theory, the method above maysuccessfully treat wrinkles as shown in FIGS. 5A, 5B and 5C. A typicalwrinkle is illustrated in FIG. 5A. FIG. 5B illustrates a threadimplanted beneath a wrinkle that is not yet hydrated. As the threadimplanted beneath the wrinkle becomes fully hydrated the surfaceappearance of the wrinkle is concurrently flattened as illustrated inFIG. 5C.

In some embodiments, the above method may be used to rejuvenate the skinof a subject in need thereof. In many of these embodiments, the threadincludes substantial amounts of non-cross linked hyaluronic acid. Insome of these embodiments, the thread includes antioxidants, vitamin Eor retinol or combinations thereof.

In some embodiments, a method of treating hair loss in a subject isprovided. A subject such as, for example, a male with typicalmale-pattern baldness is illustrated in FIG. 4A and the area where hairgrowth (with imaginary hairlines) is desired is shown in FIG. 4B. Thethread may be attached to a needle as illustrated, for example, in FIGS.1, 2A, 2B and 4C. The distal end of the needle may be inserted into oneof the hair lines as illustrated, for example, in FIG. 4C. The needlethen may traverse the area beneath the hairline of the subject and thenmay exit the skin of the subject as illustrated, for example, in FIG.4D. The needle may then be pulled distally until it is removed from thesubject such that the thread is pulled into the location previouslyoccupied by the needle as illustrated, for example, in FIG. 4E. Finally,excess thread is cut from the needle at the skin surface of the subjectwhich leaves the thread implanted as illustrated, for example, in FIG.4D.

In some embodiments, a method for treating tumors in a subject in needthereof is provided. The thread may be attached to a needle asillustrated, for example, in FIGS. 1, 2A and 2B. The distal end of theneedle may be inserted into the tumor of the subject. The needle thenmay traverse the tumor and then may exit the tumor. The needle may thenbe pulled distally until it is removed from the tumor of the subjectsuch that the thread is pulled into the location previously occupied bythe needle. Finally, excess thread is cut from the needle which leavesthe thread implanted in the tumor of the subject. In some of the aboveembodiments, the thread includes an anti-cancer agent. In someembodiments, the thread is cross linked and includes Bcl-2 inhibitors.

In an exemplary embodiment, methods of the current invention may be usedto treat pancreatic tumors. FIG. 6A illustrates a human pancreas with atumor while FIG. 6B illustrates a needle with a thread attached thereto.The pancreas may be accessed by surgery or minimally invasively methodssuch as by laparoscopy. The distal end of the needle may be insertedinto the pancreatic tumor. The needle then may traverse the pancreatictumor as illustrated in FIG. 6C and then may exit the tumor. The needlemay then be pulled distally until it is removed from the pancreatictumor such that the thread is pulled into the location previouslyoccupied by the needle. Finally, excess thread is cut from the needlewhich leaves the thread implanted in the pancreatic tumor. The processmay be repeated any number of times to provide, as illustrated in FIG.6D, a pancreatic tumor which has been implanted with a number ofthreads. In some embodiments, the thread includes an anti-cancer agent.

In some embodiments, a method for treating a varicose vein in subject inneed thereof is provided. The thread may be attached to a needle asillustrated, for example, in FIGS. 1, 2A and 2B. The distal end of theneedle may be inserted into the varicose vein of the subject. The needlethen may traverse the varicose vein and then may exit the vein. Theneedle may then be pulled distally until it is removed from the varicosevein of the subject such that the thread is pulled into the locationpreviously occupied by the needle. Finally, excess thread is cut fromthe needle which leaves the thread implanted in the varicose vein of thesubject. In some embodiments, the needle is a flexible. In otherembodiments, the thread coils when hydrated, more readily occluding thevessel.

In some embodiments, a method for nipple reconstruction is providedwhere a three-dimensional, cylindrical implant comprised of cross linkedthreads is implanted underneath the skin. The implant may includetherapeutic agents, for example chrondrocyte adhesion compounds. FIG. 7Aillustrates an implant of multiple layers of concentric coils of threadsshaped to represent a nipple while FIG. 7B shows a cross-section of theimplant of FIG. 7A. FIG. 7C illustrates how the implant of FIG. 7A couldbe used for nipple reconstruction.

In some embodiments, methods for nerve or vessel regrowth are provided.As illustrated in FIG. 8, a needle can be used to place a thread in aspecific line which could promote nerve or vessel regeneration.

EXAMPLES

The present invention is further defined by reference to the followingexamples. It will be apparent to those skilled in the art that manymodifications, both to materials and methods, may be practiced withoutdeparting from the scope of the current invention.

Example 1 Synthesis of a Cross Linked Thread

A cross linked thread of a diameter between 0.004 in and 0.006 in wasmade by forming a gel with a concentration of 5% hyaluronic acid and0.4% BDDE, by weight with the remainder comprised of water. A taperedtip nozzle with an inner diameter of 0.02 in, a syringe pressure of 20psi and a linear translation speed commensurate with the speed of gelejection from the syringe was used to extrude the gel into a threadform. However, numerous combinations of extrusion parameters that canmake a thread of the desired thickness exist. The thread was dried andthen rinsed with water which hydrated the thread, which was thenstretched during drying. Over the course of multiple rinsing and dryingcycles the overall length of the thread was increased by between about25% and about 100%. The thread made as described above will fail at atensile force of about between about 0.25 kg and about 1.50 kg and willswell in diameter by about 25% and about 100% when hydrated. It maypersist as a thread in vivo between 1 and 9 months.

Example 2 Treatment of Wrinkles of a Cadaver with Hyaluronic AcidThreads

Hypodermic needles (22 to 25 Ga) were affixed with single or doublestrands of hyaluronic acid threads, ranging from thicknesses of 0.004 into 0.008 in. Both non-crosslinked threads and threads crosslinked usingBDDE were used. The needles were able to traverse wrinkles in acadaveric head of a 50 y/o woman such as the naso-labial fold,peri-orals, peri-orbitals, frontalis (forehead), and glabellar. Theneedle was able to pull the thread through the skin such that the threadwas located where the needle was previously inserted.

Example 3 Placement of Hyaluronic Acid Threads in Dogs

Acute and chronic canine studies were performed. Hypodermic needles (22to 25 Ga) were affixed with single or double strands of hyaluronic acidthreads, ranging from thicknesses of 0.004 in to 0.008 in. Bothnon-crosslinked threads and threads cross linked using BDDE were used.In all cases, the needle was able to pull the attached thread or threadsinto the dermis. Within minutes most threads produced a visible impacton the skin surface of the animals in the form of a linear bump.

Example 4 Comparison of Tensile Strength of Different Hyaluronic AcidThreads

The tensile strength of an autocrosslinked thread of hyaluronic acid wascompared to a thread cross linked using the method of Example 1. Athread of non-crosslinked hyaluronic acid was repeatedly frozen andthawed, replicating a method of autocrosslinking hyaluronic acid (Ref.U.S. Pat. No. 6,387,413). All such samples had less tensile force atfailure than a thread made using the same extrusion parameters andcross-linked using BDDE as described above.

Finally, it should be noted that there are alternative ways ofimplementing the present invention. Accordingly, the present embodimentsare to be considered as illustrative and not restrictive, and theinvention is not to be limited to the details given herein, but may bemodified within the scope and equivalents of the appended claims. Allreferences and publications cited herein are incorporated by referencein their entirety.

What is claimed is:
 1. A dried thread comprised of (i) cross linked hyaluronic acid or a salt(s) thereof, wherein the cross linked hyaluronic acid has been cross linked with butanediol diglycidyl ether (BDDE), and (ii) non-cross linked hyaluronic acid or a salt(s) thereof.
 2. The thread of claim 1, further comprising a needle, and wherein the dried thread includes an end attached to the needle.
 3. The thread of claim 1, wherein the dried thread has a diameter of between about 0.0001 and about 0.020 in.
 4. The thread of claim 1, wherein the degree of cross linking between the hyaluronic acid or salt thereof and the cross linking agent is between about 0.01% and about 20%.
 5. The thread of claim 1, wherein the dried thread has sufficient mechanical strength for dermal implantation.
 6. The thread of claim 1, wherein the dried thread will fail at a tensile force of between about 0.1 lbs and about 5 lbs.
 7. The thread of claim 1, wherein the dried thread has an ultimate tensile strength of between about 1 kpsi and about 125 kpsi.
 8. The thread of claim 1 wherein the thread is swellable when hydrated.
 9. A dermal filler comprising the thread of claim
 1. 10. A method of treating a wrinkle comprising implanting in a subject in need thereof the thread of claim 1 beneath the wrinkle.
 11. The method of claim 10, wherein the method comprises attaching the thread to a needle; inserting the distal end of the needle through the skin surface of the subject into the dermis of the subject adjacent to the wrinkle; traversing the dermis of the subject under the wrinkle with the needle; forcing the needle through the skin surface of the subject; and cutting the thread from the needle at the skin surface of the subject.
 12. A dried thread comprised of crosslinked hyaluronic acid or a salt(s) thereof, wherein the cross linked hyaluronic acid has been cross linked with butanediol diglycidyl ether (BDDE).
 13. A dried thread comprised of crosslinked hyaluronic acid or a salt(s) thereof, wherein the crosslinked hyaluronic acid consists essentially of hyaluronic acid that has been crosslinked with butanediol diglycidyl ether (BDDE).
 14. A dried thread comprised of cross linked hyaluronic acid or a salt(s) thereof, wherein the cross linked hyaluronic acid has been cross linked with butanediol diglycidyl ether (BDDE); wherein the thread is a single strand thread; and wherein the thread is capable of being hydrated, and when hydrated will swell in diameter by about 25% and about 100%.
 15. The thread of claim 14 wherein the degree of crosslinking is between about 0.1% and about 10%.
 16. The thread of claim 15 wherein the degree of crosslinking is between about 1% and about 8%.
 17. The thread of claim 14 wherein the thread persists in vivo between 1 and 9 months after implantation in the dermis. 